Temasek Life Sciences Laboratory, National University of Singapore, Singapore.

Abstract

Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells.

(A) Venn diagram representation of upregulated genes at a minimum of two folds compared to untreated WT cells. Total RNA was extracted from cells grown to early log phase at 30°C. Probes were prepared using Low Input Quick Amp Labeling System with 100 ng of total RNA as starting material following manufacturer instruction. (B) Pearson correlation of relative protein and RNA abundance. R, Pearson correlation; P, p-value. (C) Hierarchical clustering of Log2 levels for each protein in Δcho2 and Δopi3 (left panel) and parallel coordinate plot of the nine major clusters (right panel). Major GO terms for plots i to ix are listed in .

(A) Protein expression in Δcho2 and Δopi3 compared to WT are illustrated by color-coding according to their fold change. Circle sizes are proportional to the number of proteins that were found to have similar fold change under the same cellular function. The summarized data was based on the hierarchical clustering from . (B) Expression of proteins part of the signal recognition particle (SRP), co-translational (Co-Trans) and post-translational (Post-Trans) translocation machineries. Based 2 logarithmic fold changes in protein expression were normalized to untreated WT cells. (C) Expression of proteins part of the ER-associated protein degradation (ERAD) machinery and data was normalized as in (B). Non-significant changes are denoted by “x” with p > 0.05, Student’s t-test compared to WT.

(A) Heat maps reflecting all significant lipid changes to WT. Cells were grown to early log phase at 23°C and shifted to 37°C for 2 h in selective synthetic complete media before being harvested. Lipids were extracted with chloroform:methanol mixture from lyophilized cells. Organic extraction was concentrated and analyzed by HPLC-MS. p < 0.05, Student’s t-test. (B) Cells were grown at 23°C and shifted to 37°C for 2 h in selective synthetic complete media before being prepared for TEM analysis as described in experimental procedures. N, nucleus; V, vacuole. Scale bar, 1 μm. (C) Cells were grown to early log phase at 23°C and shifted to 37°C for 2 h in selective synthetic complete media before being fixed in formaldehyde and permeabilized. Staining was performed using anti-Kar2p primary antibody followed by Alexa Fluor 488 goat anti-rabbit secondary antibody. DAPI staining marks the position of nuclei. Scale bar, 5 μm.

Cells were grown to early log phase at 23°C and shifted to 37°C for 30 min (sec63-1) or 2 h in selective synthetic complete media in the absence (A) or presence (B) of 1 mM choline before being pulse-labeled at 37°C with L-[35S]-methionine/cysteine for 5 min followed by a chase at the indicated times. Immunoprecipitated proteins using anti-CPY or anti-Gas1p were resolved by SDS-PAGE and visualized by phosphoimager analysis.

The UPR compensates protein quality control defect during lipid imbalance

Cells were grown to early log phase at 23°C and shifted to 37°C for 2 h in selective synthetic complete media in the absence (A) or presence (B) of 1 mM choline before being pulse-labeled at 37°C with L-[35S]-methionine/cysteine for 10 min followed by a chase at the indicated times. Immunoprecipitated proteins using anti-HA were resolved by SDS-PAGE and quantified by phosphoimager analysis. Data plotted is the mean ± SD of three independent experiments. *p < 0.02, Student’s t-test.